1. Field of the Invention
This invention relates to an immunodiagnostic assay method and kit to rapidly detect oral cariogenic bacteria, i.e., mutans streptococci, Lactobacillus sp. and Actinomyces sp., in human dental plaque, saliva and oral rinse samples.
2. Description of the Prior Art
Certain species of oral bacteria have been associated with dental caries in humans (1). The presence of mutans streptococci, which includes Streptococcus mutans and Streptococcus sobrinus, is highly correlated with dental caries and this has been widely addressed in the dental literature (1-23). Streptococcus mutans is considered the foremost bacterial species associated with the development of human dental caries (12). Various serotypes (serovars) of Streptococcus mutans have been described (17). Lactobacillus sp. (2, 3, 13, 14, 18-24) and Actinomyces sp. (19, 23-26) have also been associated with human dental caries. Ebersole has described a SEROLOGICAL METHOD FOR THE IDENTIFICATION OF MICROORGANISMS in U.S. Pat. No. 4,458,014 specifically for the identification of diseases of the mouth. Chen et al. have described in U.S. Pat. No. 4,866,167 a DETECTION OF HUMAN ORAL CELLS BY NUCLEIC ACID HYBRIDIZATION to detect Streptococcus mutans and other bacterial species. The methods of both Ebersole and Chen et al. are technically complex, time consuming and are not rapid.
Toshitsugu et al. European patent 0 496 345 A1 have described a METHOD FOR DETECTING AND QUANTIFYING CARIOGENIC BACTERIA. There are several important differences between their invention and the invention described herein. Toshitsugu et al. do not strip off antibodies present on bacteria prior to reacting bacteria with antibodies. They disperse their sample containing Streptococcus mutans to "an optimal buffer solution suitable for the antigen-antibody reaction . . . " The invention described herein uses a buffer that is not suitable for the antigen-antibody reaction and, to the contrary, strips off antibodies from oral bacterial antigens. Toshitsugu et al. use conventional millipore membrane filters activated by vacuum (suction), centrifugation, or syringe pressure; the invention described herein uses a flow-through device which filters passively by gravity or wicking action. Filtration to Toshitsugu et al. means filtration under pressure using a syringe, suction/vacuum filtration, or centrifugation filtration; the invention described herein never uses any of those active types of filtration. Toshitsugu et al. have no negative control built into their system; they react the entire surface of the membrane filter. The invention described herein reacts in only a spot on the filter surface leaving the surrounding area as a negative control which is not possible with their test. Their test also does not exclude the possibility that the peroxidase they detect may be of human or microbial origin. The test of Toshitsugu et al. is not rapid. Their test always takes more than one hour--usually from 1 to 18 hours, or longer. The test specified in most of their examples takes over 2 hours; the test described herein can be performed in 5 minutes. Additionally, as mentioned in their example 4, they incubate twice at 37.degree. for one hour for their antigen-antibody reaction and indirect labeling; the invention described herein never requires an incubator. In all their examples, they use a spectrophotometer to read color development in fluid; the test described herein never uses a spectrophotometer to read color development; in the test described herein color development is done visually against a standard and is not read in fluid. There are some other lesser differences, but nevertheless important between their patent and the one described herein. In some cases, they disperse the plaque sample by sonication as in their example 13; sonication is never used in the invention described herein.
Their filter is placed in a separate well for color development; the filter is not removed for color development with the invention described herein. Also in the invention described herein, polyclonal antibodies are absorbed to make them specific; Toshitsugu et al. make no mention of absorption and mention that specificity of polyclonal antibodies is a problem. Lastly, they have a filter membrane air drying step; the filter in the invention described herein is never dried in the air or removed for measurement.
The use of antisera to mutans streptococci, Lactobacillus sp. and Actinomyces sp., separately or in combination, has not been reported as part of a simple, rapid, chairside assay. The rapid detection of these cariogenic bacteria assists in the accurate diagnosis and prediction of active dental caries where other methods are limited and at a rate faster than now available through Chen et al., Ebersole or Rosenberg et al. U.S. Pat. No. 4,976,951, DENTAL CARIES DIAGNOSTIC AND LOCALIZATION TECHNIQUE.
Previous methods can detect Streptococcus mutans levels in 48 hours using dental plaque (U.S. Pat. No. 3,746,624) or saliva (Dentocult SM Strip Mutans, Vivacare diagnostic line/VIVADENT, manufactured by Orion Diagnostica, Espoo, Finland). These older methods are considered too slow, particularly by dentists seeking immediate answers for patients in their care and by persons who are traveling to distant locations where dental services are difficult to obtain. What is needed is a simple to operate assay to detect cariogenic bacteria that can be developed and read in less than an hour, requires no expensive equipment and can be performed preferably in five minutes or less.